Effect of Cigarette Smoking on Human Gingival Mucosa-Histological andMorphometric StudyGhada Galal Hamam * and Dalia Alaa El-Din Aly El-WaseefDepartment of Histology and Cell Biology, Faculty of Medicine, Ain Shams University, Cairo, Egypt*Corresponding author: Hamam GG, Department of Histology and Cell Biology, Faculty of Medicine, Ain Shams University, Cairo, Egypt, Tel: 0020 01003960601; E-mail: [email protected]
Received date: July 30, 2018; Accepted date: August 14, 2018; Published date: August 20, 2018
Introduction: Cigarette Smoking is considered the most preventable cause of diseases and premature deaths inthe history of mankind. It is associated with high levels of morbidity and mortality all over the world.
Aim of the study: To study the light and electron microscopic structure of clinically normal gingival mucosa innonsmoker versus smoker adult males.
Subjects and methods: Eighteen adult males (volunteers) were divided according to the intensity and durationof smoking (pack year index) into three groups. Group I (non-smokers control), group II (pack year index 1-9) andgroup III (pack year index >10). Specimens from the gingival mucosa were obtained and processed for properhistological techniques. Paraffin sections were stained with H&E, Mallory`s trichrome stain andimmunohistochemical reaction for caspase-3. Transmission electron microscopic study, morphometric and statisticalanalysis were also performed.
Results: Basal cell hyperplasia, increase thickness of epithelium, apoptosis of epithelial cells, and inflammatorycellular infiltration in the lamina propria, desmosomal disruption, increase glycogen granules and melanin pigmentswere noticed in group II and were more increased in group III.
Conclusions: Clinically normal gingival mucosa of cigarette smokers shows light and electron microscopicstructural changes that are directly related to the pack year index of the individual.
IntroductionTobacco smoking is a global problem of civilization; with the
estimated number of tobacco smokers about 1.3 billion. Statistically,the life expectancy of smokers is 15 years less than that of non-smokers[1]. Cigarette smoke increases the susceptibility to oral mucosalinfection. It is a well-recognized risk factor for periodontitis, oralcandidiasis and oral leukoplakia [2,3].
The use of tobacco and its related products are still very prevalent,although it is an established fact that they are one of the leadingcausative agents for oral cancer. Oral cancer is one of the mostdebilitating diseases affecting men [3]. It was reported that almost263,020 oral cavity cancers and 127,654 oral cancer deaths occurworldwide each year. Individuals at high risk of developing oral cancerare mainly older, males, heavy tobacco smokers and alcohol users, andthose with poor diet and low socioeconomic status [4].
Tobacco smoke contains more than 4000 substances that influencethe human body, about 60 of which are known carcinogens. Moreover,it is reported that Tobacco smoke affects the normal function of theimmunological system, increasing the risk of respiratory infections,which may also contribute to the development of malignancies. Duringsmoking one cigarette, the smoker absorbs about 500 mg of smoke,
about 65% of it is nitrogen and oxygen, and 35% consists ofbiologically active substances [1].
The epithelium of the oral mucosa is the first tissue that encountersdiverse toxic substances in cigarette smoke [2]. Although smokinginduces similar changes in gene expression in the oral and bronchialepithelium concerning carcinogenesis and the activity of selectedchemopreventive agents and targeted anticancer drugs, and despite theeasy accessibility of the oral epithelial cells-which give insights intotobacco-induced molecular changes in the oral cavity as well as thebronchial epithelium, still the effects of nicotine on epithelial cells ofthe respiratory tract are well documented [5], but the epithelium of theoral cavity has not been so well explored.
The oral epithelium is a stratified squamous epithelium (keratinizedor parakeratinized in areas subjected to mastication as the gingiva andpalate). The layers of the oral epithelium represent a continuousprocess of maturation where the superficial keratinized cells areconstantly shed and replaced from below. There is a balance betweenthe dying and proliferating cells. The process of keratinization isconsidered a physiological cell death, occurring by the mechanisms ofapoptosis. However, in apoptosis, cells degenerate into apoptoticbodies, while keratinocytes reach the final stage of differentiationbecoming flat, filled with closely packed keratins, and surrounded by acornified envelope of the mature cell. Apoptosis occurs through twopathways; the extrinsic pathway leading to activation of procaspase 8
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ISSN: 2157-7099
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DOI: 10.4172/2157-7099.1000517
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J Cytol Histol, an open access journalISSN: 2157-7099
then caspase 3which causes self-destruction of cell, and the intrinsic(mitochondrial) pathway where factors inside the cell can induceapoptosis such as DNA damage, oxidative stress and reactive oxygenspecies. These factors activate procaspase 9 then caspase 3. Caspase 3 isinvolved in proteolysis of important proteins and activation ofendonucleases that cut DNA into fragments [1].
Caspases are cysteinyl aspartate-specific proteases that play a pivotalrole not only in the induction of apoptotic cell death but also in theinflammatory responses against microbial infection. Caspases aredivided into three functional groups: apoptosis induction (Caspase-2,3, 6, 7, 8, 9, and 10), inflammatory responses (Caspase-1, 4, 5, 11, and12) and differentiation (Caspase-14) [6].
For the previous reasons, this study was designed to study theinfluences of cigarette smoke on the structure of clinically normalgingival mucosa in healthy men volunteers.
Subjects and methodsThe study was conducted on 18 volunteer male patients who
attended the First Dental Center of Egypt (Confi Dent) private clinic inthe period from January 2017 till September 2017. This study wasapproved by the Research and Ethics Committee of Ain ShamsUniversity. All 18 participants were fully informed about the objectivesof the study and had received printed information and signed a writtenconsent according to the guideline and regulation of the EthicsCommittee.
Methodology
Every patient was subjected to the following:
Careful history takingThe aim of the study was explained to each subject and if consenting
to participate, history was taken, and a questionnaire was filled.History of smoking, alcohol use, medications and systemic diseaseswas reported. Family history of oral lichen planus and oral cancer infirst degree relatives was also reviewed. The level of oral hygiene wasestablished as a function of daily brushing, evaluated as good if thepatient brushed three or more times per day, moderate if brushing wasonce or twice a day, and poor if brushing was infrequent or null. Thecause of attending the dental clinic was also reviewed. The significantsystemic diseases were hypertension diabetes, hypercholesterolemia,and chronic impairment of renal function and psychiatric status asdepression or stress for long period.
For smoking, it was first established if the patient was a smoker ornot, and when affirmative, the type and quantity of tobacco smokedper day and the duration of smoking were assessed. It was reportedthat smokers are those who consumed at least one cigarette per day fora minimum period of one year [7].
The type of consumed cigaretteThe commercial type of cigarette was fixed in all participants. This
type contained 1 mg of nicotine and 15 mg of tar in each cigarette.
Examination of the oral cavityAll participants underwent an oral clinical examination which was
performed by experienced stomatologist, through visual inspection
and palpation of oral mucosa and teeth. Examination was based on thecriteria for the Visual detection of oral mucosal lesions and the WorldHealth Organization (WHO) criteria.
Criteria for patient's selectionThe inclusion criteria: Male volunteers aging from 25-45 years;
available at the time of survey; and willing to give written consent wereincluded in this study.
The exclusion criteria: Patients with the following criteria wereexcluded from the study:
• Patients under medication as corticosteroids or chemotherapeuticdrugs.
• Patients diagnosed with cancers, systemic diseases, chronicalcoholism, recent viral infection and drug addiction were alsoexcluded.
• Patients with prosthetic devices (removable or fixed)• Patients with untreated periodontal diseases.• Occasional smokers were excluded from the study as well as those
who were previously smokers then stopped smoking.• Pateints with poor oral hygiene.• Cigar, pipe and shisha smokers were also excluded.
Sample size calculation was kept at minimum required to providethe power sufficient for statistical comparisons.
Study design and groupingPatients, who were classified as non-smokers, had no history of
tobacco smoking at all. For ethical reasons, smokers were informedabout the potentially negative effects of smoking on health and wereencouraged to cease smoking.
On observation of any suspicious lesion after examination of theslides, the corresponding patient was informed and advised to befollowed up by routine biopsy every 6months to be referred tooncologist upon need.
Eighteen male participants were included in this study and wereclassified according to pack year index into three groups:
Group I: non-smokers (n=6)
Group II: pack year index 1-9 (n=6)
Group III: pack year index > 10 (n=6)
A pack year is a unit for measuring the number of cigarettes aperson has smoked over a certain period of time. It is calculated bymultiplying the number of packs of cigarettes smoked per day by thenumber of years of smoking [8,9].
The equation used
Number of pack years=(Number of cigarettes per day/20) × numberof years of smoking
of years of smoking (1 pack has 20 cigarettes)
For example: a person who has smoked 15 cigarettes a day for 40years had a (15/20) × 40=30 pack-year smoking history.
Sample collection and processingLight microscopic study: A 3 mm punch biopsy was obtained from
the outer gingival mucosa behind the right wisdom tooth of the lower
Citation: Hamam GG, El-Waseef DAEA (2018) Effect of Cigarette Smoking on Human Gingival Mucosa-Histological and Morphometric Study. J CytolHistol 9: 517. doi:10.4172/2157-7099.1000517
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Number of pack years=(Number of cigarettes per day/20) x number
jaw from each participant. The biopsy was obtained from clinicallynormal gingival mucous membrane using Kai medical disposablebiopsy punch, single use (Kai industries Co. ltd. 1110 Oyana, Seki City,Gifu Pref., 501-39992, Japan) under Mepecaine-L local anesthesiapurchased from Alex For Chemical Industries and Drugs. Specimenswere immediately fixed in 10% neutral buffered formalin and wereprocessed into paraffin blocks. Five μm thick sections were cutperpendicular to the epithelium. Then they were stained with H and Eand Mallory’s trichrome stain. Other sections were cut on positivelycharged slides to be subjected to caspase-3 immunohistochemical stain[10].
Immunohistochemical study: Caspase-3 (ready to use) was used fordetection of apoptosis in the epithelium of gingival mucosa. Paraffinsections of the mucosa and of a positive control (tonsils) wereprocessed. Negative control was processed according to the sameprotocol, except for the use of the primary antibody [10]. Positiveexpression of Caspase-3 was detected as brown cytoplasmic reactionwith some nuclear staining. The kit was supplied by Lab VisionCorporation. Kato Road, Fremont, USA.
Transmission electron microscopic study: One specimen from eachgroup was immediately fixed in glutaraldehyde and was processed fortransmission electron microscopic (TEM) study. Each specimen wascut into small pieces (1 mm3) and rapidly fixed in 2.5% glutaraldehydefor 24 h. Then they were processed and embedded in Epon resin.Ultrathin sections (50 nm thickness) were cut and mounted on coppergrids [10]. Specimens were examined and photographed using a JEM1200 EXII, JEOL, Tokyo, Japan transmission electron microscope(TEM) at the Faculty of Science, Ain Shams University.
Morphometric and statistical study: Samples were examined usingLeica DM2500 microscope with built in camera (Wetzlar, Germany).All images were digitally acquired using an image analyzer Leica Q winV.3 program (Wetzlar, Germany) installed on a computer in theHistology and Cell Biology department, faculty of Medicine, AinShams University. Specimens from all groups were subjected tomorphometric study. Measurements were taken from three differentslides obtained from each specimen. Five different non-overlappingfields were examined from each slide at objective lens ×10 to measurethe first three parameters and ×20 to measure the area percentage:
• Epithelial thickness. It was measured from the surface of theepithelium to the basement membrane by a line perpendicular tothe surface.
• Length of epithelial ridges.• Ratio of basal lamina length to epithelial surface length.• Area percentage of collagen fibers.• Area percentage of caspase-3 reaction.
All values were presented as mean ± SD. The differences among thegroups with respect to all measured data parameters were statisticallyanalyzed using one-way analysis of variance and the post-hoc testusing SPSS program version 19 (IBM Corporation, Somers, New York,USA). The calculations were considered significant if P value was lessthan 0.05.
Results
Light microscopic (LM) resultsExamination of H and E stained sections from the gingival mucosa
(gums) of non-smokers (group I) showed the masticatory mucosa
formed of gingival epithelium (stratified squamous non-keratinizedand para-keratinized epithelium) and lamina propria. The laminapropria consisted of a papillary layer formed of loose vascularconnective tissue and a reticular layer formed of denser connectivetissue. Gingival epithelium interdigitated with connective tissueforming interconnected rete ridges that were seen separated byconnective tissue papillae (Figure 1A). The gingival epithelium wasformed of stratum basale which appeared as a layer of small cuboidalcells resting on the basement membrane. Their nuclei appeared closelypacked. Stratum spinosum appeared as many layers of polygonal cells,with central rounded vesicular nuclei. Stratum superficial was seen asflattened superficial cells with flattened nuclei. In the Para keratinizedareas, the superficial cells did not lose their nuclei (Figure 1B).
In group II, apparently increased thickness of the epithelium withprominent epithelial ridges was noticed in some areas (Figure 1C).Proliferation of cells of basal layers was noticed. Most cells of stratumspinosum and superficial appeared vacuolated with pyknotic nuclei.Localized areas of proliferation of cells of stratum superficial (epithelialnests) were sometimes noticed (Figure 1D). Intra epitheliallymphocytes, inflammatory cells in the papillary dermis and dividingcells in stratum basale were frequently seen (Figure 1E).
In group III, apparently increased thickness of the epithelium andprominent epithelial ridges were frequently noticed (Figure 1F).Proliferation of cells of stratum basale, vacuolation of cells of stratumspinosum with irregular pyknotic nuclei, karyorrhexis and karyolysiswere frequently noticed. Most cells of stratum superficial appearedvacuolated. Some of them appeared with rounded nuclei. Karyorrhexisand karyolysis of most nuclei were seen (Figure 1H). Intra epitheliallymphocytes and mononuclear cellular infiltration in the papillarylayer of dermis under the epithelium were also seen (Figure 1G).Increased vascularity of papillary layer of dermis and congestion ofblood vessels were also noticed (Figure 1I).
With examination of Mallory’s trichrome stained sections in groupI, collagen fibers in the papillary layer appeared as fine interlacingbundles. In the reticular layer, collagen fibers were seen arranged incoarse wavy bundles running in different directions. Most bundleswere of uniform diameter (Figure 2A and 2B). In group II, irregularshort fine collagen fibers were seen in the papillary layer. In thereticular layer, thin irregular disorganized collagen bundles were seen(Figure 2C and 2D). In group III, collagen fibers were seen thin, shortdisorganized, fragmented and scanty in both the papillary and thereticular layer (Figure 2E and 2F).
Immunohistochemical resultsExamination of sections from group I showed negative caspase-3
reaction in cells of stratum basale and spinosum. Few cells withpositive immune reaction were noticed in stratum superficial (Figure2G). In group II, caspase-3 positive immune reaction was seen in thecytoplasm of cells of stratum spinosum and superficial. Negativereaction was seen in cells of stratum basale (Figure 2H). In group III,many caspase-3 positive cells were seen in cells of stratum basale,spinosum and superficial (Figure 2I).
Transmission electron microscopic resultsExamination of ultrathin sections of human gingival mucosa from
group I showed the layers of the oral epithelium (stratified squamousepithelium) formed of stratum basale resting on the basementmembrane that separated keratinocytes from underlying connective
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Hamam GG, El-Waseef DAEA (2018) Effect of Cigarette Smoking on Human Gingival Mucosa-Histological and Morphometric Study. J CytolHistol 9: 517. doi:10.4172/2157-7099.1000517
tissue (Figure 3A). Keratinocytes of stratum spinosum appearedpolygonal in shape with large, central euchromatic nuclei.Desmosomes were seen between the cells (Figure 3B). Cells of thedeeper layers of stratum superficial were seen slightly flattened withoval nuclei. Glycogen granules were seen in their cytoplasm (Figure
3C). Superficial cells of stratum superficial were seen more flattened(squamous) with flattened nuclei. Fine micro-ridges were also noticedon the free surface of cells. Desmosomes connected adjacent cellmembranes.
Figure 1: H and E stained sections. (A and B) Group I: (A) the gingival mucosa is formed of stratified squamous para-keratinized epithelium.The lamina propria consists of papillary layer (P) formed of loose connective tissue. The reticular layer (R) is formed of denser connectivetissue. Connective tissue papillae interdigitate with epithelial ridges (pegs) () Inset: blood vessels in the papillary layer. (B) The gingivalepithelium is formed of stratum basale (B) which is a layer of small cuboidal cells resting on the basement membrane. The nuclei appearclosely packed. Stratum spinosum (S) appears as many layers of polygonal cells with central rounded nuclei. Inset: stratum superficial in thepara-keratinized area appears as flattened superficial cells with flattened nuclei. (C-E) Group II: (C) Area of apparent increase thickness of theepithelium (E), prominent epithelial ridges, numerous dermal papillae and areas of localized cellular aggregation at the surface (↑) are seen.Inset: blood vessels in the papillary layer. (D) area of proliferation of basal layer (B) is noticed. Most cells of stratum spinosum and are seenvacuolated with pyknotic nuclei (↑). Upper inset: superficial vacuolated cells (↑) are seen in the para-keratinized epithelium. Lower inset:localized area of proliferation of cells of stratum superficial (epithelial nest). (E) showing intra epithelial lymphocytes (↑) and inflammatorycells in the papillary dermis (*). (F-I) Group III: (F) Areas of apparent increase in thickness of the epithelium (E) and prominent epithelialridges can be seen. (G) Proliferation of cells of stratum basale (B), vacuolation of cells of stratum spinosum with irregular pyknotic nuclei ()and intra-epithelial lymphocytes (↑) are seen. Mononuclear cellular infiltration (*) can also be seen in the papillary layer of dermis under theepithelium. Inset: karyorrhexis (↑) and karyolysis () of nuclei of cells of stratum spinosum. (H) Most cells of stratum superficial are vacuolated.Karyorrhexis (↑) and karyolysis () of some nuclei are seen. Some superficial cells show rounded nuclei (↑↑). Nest of squamous epithelium isseen (∆). (I) increase vascularity of papillary layer of dermis and congestion (*) of some vessels can be seen. A, C and F (X 100); B, D, E, G, Hand I (X 400); Insets of A, B, C and D (X 400); Inset of G (X 1000).
Citation:Hamam GG, El-Waseef DAEA (2018) Effect of Cigarette Smoking on Human Gingival Mucosa-Histological and Morphometric Study. J CytolHistol 9: 517. doi:10.4172/2157-7099.1000517
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Figure 2: (A-F) Mallory stained section. (A and B) Group I: (A) Collagen fibers in the papillary layers appear as fine interlacing bundles. (B) Inthe reticular layer, collagen fibers are arranged in coarse, wavy bundles running in different directions. Most bundles are of uniform diameter.(C and D) Group II: (C) showing irregular short fine collagen fibers in the papillary layer. (D) In the reticular layer thin irregular disorganizedcollagen bundles are seen. (E and F) Group III: (E) Showing thin short and scanty collagen fibers in the papillary layer. (F) In the reticularlayer, thin disorganized fragmented and scanty collagen fibers are seen. (G-I) Caspse-3 immune reaction. (G) Group I: negative caspase-3reaction is seen in cells of stratum basale and spinosum. Inset: positive immune reaction in cells of stratum superficial (↑). (H) Group II:negative caspase-3 reaction is seen in cells of stratum basale (B) and positive reaction is seen in the cytoplasm of cells (↑) of stratum spinosum.Inset: positive immune reaction in cells of stratum superficial (). (I) Group III: many caspase-3 positive cells (↑) are seen in cells of stratumbasale and spinosum. Inset: caspase-3 positive reactions are seen in cells () of stratum superficial. A-F (Mallory`s trichrome stain X 400); G- I(Caspase-3 × 40); Insets of G, H and I × 400.
Detached cells were sometimes noticed (Figure 3D). In group II,cells of stratum basale were seen with euchromatic nuclei and restingon basement membrane separating them from underlying connectivetissue. Desmosomes were seen between adjacent cells (Figure 4A).Most cells of stratum spinosum appeared with intracellular vacuoles(Figure 4B), disruption of desmosomal plaques and widening of theintercellular spaces. Apoptotic cells were also seen containing no nuclei(Figure 4C).
The deep layers of stratum superficial showed large amount ofintracellular electron dense glycogen granules (Figure 4D). Superficialcells of stratum superficial were seen flattened with fine micro-ridges
on their free surface. Some nuclei appeared with disturbed unclearmembrane and chromatin arrangement (Figure 4E). In group III, cellsof stratum basale were seen resting on irregular basement membrane.
Electron dense ellipsoid granules were also seen. Wide intercellularspaces and faint nuclei were frequently noticed (Figure 5A).Intracellular electron dense glycogen granules, wide intercellular spaceand disruptions of desmosomal attachment plaques were frequentlyseen (Figure 5B). Irregular nuclear envelope, clumped chromatin andperinuclear glycogen granules were seen in most cells of stratumsuperficial. Disturbed micro-ridges were also noticed on the freesurface of the squamous cells (Figure 5C).
Citation: Hamam GG, El-Waseef DAEA (2018) Effect of Cigarette Smoking on Human Gingival Mucosa-Histological and Morphometric Study. J CytolHistol 9: 517. doi:10.4172/2157-7099.1000517
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Figure 3: (A-D) Group I: (A) Stratified squamous lining of the human gingival mucosa. A continuous basement membrane (↑) separateskeratinocytes in the stratum basale (B) from underlying connective tissue. (B) Keratinocytes in the stratum spinosum are polygonal in shapewith large, central euchromatic nuclei with two nucleoli. Desmosomes (↑) are seen between the cells. (C) Cells of the deeper layers of stratumsuperficial are seen slightly flattened with euchromatic nuclei (↑) and glycogen granules () in their cytoplasm. (D) Superficial cells of stratumsuperficial are more flattened (squamous) with flattened nuclei. Fine micro-ridges are seen on the free surface of the squamous cells ().Desmosomes connect adjacent cell membranes (↑). Beginning of detachment of cells is seen (*). A, B, C and D (TEM × 1500).
Citation:Hamam GG, El-Waseef DAEA (2018) Effect of Cigarette Smoking on Human Gingival Mucosa-Histological and Morphometric Study. J CytolHistol 9: 517. doi:10.4172/2157-7099.1000517
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Figure 4: (A-E) Group II: (A) cells of stratum basale are seen with euchromatic nuclei (N). irregular Basement membrane (↑) separates themfrom underlying connective tissue. Desmosomes () are seen between adjacent cells. (B) indented nucleus (↑) and intracellular vacuoles () areseen in cells of stratum spinosum. (C) apoptotic cell, disruption of desmosomal plaques and wide intercellular spaces are seen between cells ofstratum spinosum. (D) The deep layers of stratum superficial show large amount of intracellular electron dense glycogen granules (↑). (E)Superficial cells of stratum superficial are seen flattened with fine micro-ridges on their free surface (). A nucleus (↑) with disturbed nuclearmembrane and chromatin arrangement is seen. A, B, D and E (TEM × 1500); C (TEM X 2500).
Histomorphometric resultsIn the present study, 18 individuals were included. The age range of
non-smokers was 28-42 years with a mean of 32.8 ± 6.2 years; the agerange of smokers in group II was 26-38 years with a mean of 32 ± 4.5years and the age range of smokers in group III was 28-45 years with amean of 36.3 ± 11.1 years (Table 1).
In group II, the range of daily cigarette consumption was 5-15cigarettes (9.6 ± 7.4), for 4-15 years (9.2 ± 4.1) with range of pack yearindex 1-7.5 (4.5 ± 2.8). In group III, the range of daily cigarette
consumption was 12-30 (20.3 ± 9.5) for 10-30 years (19 ± 10) withrange of pack year index 10-30 (16.5 ± 11.3) (Table 1).
The mean thickness of both the epithelium and the epithelial ridgeswere significantly increased in groups II and III compared to controlgroup. The percentage ratio of basal lamina length to epithelial surfacelength was significantly increased in groups II and III compared tocontrol group. These parameters showed no significant differencebetween groups II and III (Table 2 and Figure 6).
Citation:Hamam GG, El-Waseef DAEA (2018) Effect of Cigarette Smoking on Human Gingival Mucosa-Histological and Morphometric Study. J CytolHistol 9: 517. doi:10.4172/2157-7099.1000517
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Figure 5: (A-C) Group III: (A) Irregular basement membrane (↑) separates keratinocytes in the stratum basal (B) from underlying connectivetissue. Cells are seen with electron dense melanin granules. Wide intercellular spaces (*) and faint nuclei are seen. (B) intracellular electrondense glycogen granules, wide intercellular space (*), disruptions of desmosomal attachment plaques between cells of stratum spinosum areseen. (C) Stratum superficial is seen as flattened cells with flattened nuclei. Irregular nuclear envelope (↑), clumped chromatin and perinuclearglycogen granules () are seen. Disturbed micro-ridges can be seen on the free surface of the squamous cells (). A, B and C (TEM × 1500).
The mean area percentage of collagen fibers in the dermis wassignificantly decreased in groups II and III compared to control group.Group III also showed significant decrease compared to group II(Table 2 and Figure 6).
There was a significant increase in the mean area percentage ofcaspase-3 reaction in the epidermis in groups II and III compared tocontrol group. A significant increase was also noticed in group IIIcompared to group II (Table 2 and Figure 6).
Group I Group II Group III
Number of cases 6 6 6
Age 32.8 ± 6.2 32 ± 4.5 36.3 ± 11.1
Daily consumption - 9.6 ± 7.4 20.3 ± 9.5
Duration-year - 9.2 ± 4.1 19 ± 10
Pack Year Index 0 4.5 ± 2.8 16.5 ± 11.3
Table 1: History and mean ± SD of smoking habits characteristics in different groups.
Significant increase compared to control*Significant change compared to other groups
Table 2: Mean ± SD of epithelial thickness, length of epithelial ridges, basal lamina/epithelial surface length, area% of collagen fibers and area% ofcaspase-3 positive immune reaction in different groups.
Citation:Hamam GG, El-Waseef DAEA (2018) Effect of Cigarette Smoking on Human Gingival Mucosa-Histological and Morphometric Study. J CytolHistol 9: 517. doi:10.4172/2157-7099.1000517
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Figure 6: The mean thickness of epithelium, the mean thickness of epithelial ridges, the ratio of basal lamina length/epithelial surface length,the mean area percentage of collagen fibers in dermis and Caspase-3 reaction in different groups.
DiscussionThe first exposure of cigarette smoke is experienced by the oral
tissues, and its chemical components are rapidly absorbed andcirculate in the blood stream [11]. So, we sought to investigate theeffects of whole cigarette smoke on the structure of normal humangingival mucosa in healthy males. Women were not included in thisstudy, to avoid cellular changes that occur during menstruation, aftermenopause and in pregnancy, as previously reported by some authors[12].
In the current study, pack year index was used to evaluate the effectsof smoking as it was reported that pack year index describes the degreeof exposure to tobacco [8].
In the current study, basal layer hyperplasia was noticed in groups IIand III and the mean thickness of gingival epithelium was significantlyincreased in both groups, compared to control group. This wasexplained by some authors who reported that cigarette smoke directlyactivates epithelial cell proliferation [13,14] and causes basal layerhyperplasia in the mucosa of pharynx, larynx and tongue of rats [13].Proliferation of basal cell layer of the epithelium might be a protectivemechanism against irritation of smoking which leads to proliferativeresponse of basal stem cells.
In the current study, elongation of epithelial ridges and increaseratio of basal lamina length to epithelial surface length were noticed ingroups II and III. It was reported that the length of the basal lamina is
directly related to the overall number of basal cells that participate inepithelial renewal. The longer the basal lamina below the epithelialsurface; the greater is the number of the basal cells that can entermitosis [15]. It was also reported that tobacco components stimulateproduction of proinflammatory cytokines, which could induceepithelial cells proliferation and migration to help epithelizationduring wound healing [16]. Also, it was previously reported thatnicotine, a component of tobacco smoke, significantly stimulates cellgrowth [17]. These facts could be a good explanation for the thickeningof epithelium observed in groups II and III in the current study.
In the current study, localized areas of epithelial hyperplasia (cellnest) were noticed in groups II and III. It was reported that remnant ofdental lamella is called small nests of squamous epithelium. Theseepithelial nests may proliferate and undergo degeneration forminggingival cyst, which is premalignant [18]. Metaplasia represents anadoptive and reversible response to persistent injury of the epitheliumcaused by chronic infections. It results from a reprogramming ofepithelial stem cells that begin to differentiate into new cell lineage[19].
Cigarette components as nicotine, carbon monoxide, and reactiveoxygen species can penetrate the cells and induce DNA damage,leading to various mutations [16]. In groups II and III, various changeswere noticed in the nuclear structure ranging from irregular nuclearmembrane, clumped chromatin, pyknosis, karyolysis and karyorrhexis.It was reported that nuclear damage triggers impairment of p53/p21
Citation:Hamam GG, El-Waseef DAEA (2018) Effect of Cigarette Smoking on Human Gingival Mucosa-Histological and Morphometric Study. J CytolHistol 9: 517. doi:10.4172/2157-7099.1000517
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pathways. Stem cells need regulated amount of p21which is a cell cycleinhibitor. It has an essential role in maintaining stem cells quiescence.In the absence of p21, stem cells fail to maintain quiescence resultingin hyperproliferation and expansion of progenitor cell pools resultingin exhaustion of stem cell pool [20].
It was reported that normal renewal of the oral epithelium is theresult of equilibrium between rate of proliferation and rate of cell death[21]. Very little is known about the possible impact of tobacco smokeon the apoptosis of epithelial cells in the oral cavity, and observationsvary, from no changes on the level of apoptosis to increased levels ofhypodiploid cells in the epithelium of smokers through the inhibitionof apoptosis under influence of nicotine. However, these observationsare based on in vitro cultured keratinocytes and animal models [1]. Inthe current study, a significant increase in the mean area percentage ofcaspase-3 expression was noticed in smokers of groups II and III,meaning that smoking increased the rate of cell death. However, ourlight microscopic and morphometric results showed a significantincrease in the thickness of the epithelium and in the proliferation ofthe basal layer in groups II and III. The previous findings mean thatthe proliferative capacity of the epithelium under the effect of smokingmarkedly exceeded the rate of cell death. Added to the effect ofcarcinogenic products in cigarette smoke, all these factors put thegingival epithelium at a high risk of developing cancers. It was alsoreported that apoptosis progression relies on the interaction betweenpro-survival factors and pro-apoptotic factors, and this balance decideswhether a cell under stress lives or dies. Pro-survival proteins caninhibit the apoptotic cytoplasmic caspases. Injury to superficial airwayepithelium is normally accompanied by mitotic activity in theremaining cells and rapid restoration of the denuded surface. Tobaccosmoke is a potent stimulus of DNA damage by oxidant injury. Tobaccosmoke may affect the expression and activity of proteins associatedwith cell proliferation and cell apoptosis thus altering physiologicalreparative mechanisms [22].
An important property of epithelium is its ability to function as abarrier. Desmosomes or maculae adherens are modifications ofadjacent membranes of cells to ensure proper barrier [23]. In thecurrent study, ultra-structural analysis of gingiva of groups II and IIIshowed disruption of desmosomes between cells of stratum spinosum.This might be explained by the fact that cigarette smoke increasescytokines which destroy the cell-cell interaction proteins (e.g. E-cadherin) [16].
In electron microscopic examination of groups II and III,intracellular glycogen was noticed in cells of stratum spinosum andsuperficial of the gingival epithelium. This might explain thevacuolation seen in H and E stained sections in the epithelium of thesetwo groups.
Ellipsoid electron dense granules mostly melanin granules werenoticed in cells of stratum basale in group III. Other authors alsoreported increased melanin granules in smokers. They attributed thisto the development of smokers' melanosis [24].
Intraepithelial lymphocytes were noticed in groups II and III. It wasreported that intraepithelial lymphocytes are found in epithelialmucosal linings. Upon encountering antigens, they immediatelyrelease cytokines and cause killing of infected target cells. They act atepithelial border to eliminate injured and infected cells. It was reportedthat in mucosal cell surface, T cell receptors are mainly of γ and δchains, not α and β chains. The γ /δ T cells are characterized by being a
bridge between the innate and adaptive immune system. They act asthe first line of defense against microbes [25].
In the current study, inflammatory cells were noticed in laminapropria of groups II and III. Similar finding was also noticed by someauthors [26]. It was reported that tobacco components produceproinflammatory cytokines that cause chronic inflammation.Cytokines are inflammatory factors that are normally secreted due totissue injury to promote repair process. Till now, the cytokines thathave been found to increase after exposure to cigarette smoke are theinterleukins (IL)-1, IL-6, IL-8, IL-10, tumor necrosis factor-α, as well astransforming growth factor-β, granulocyte-macrophage colony-stimulating factor and monocyte chemoattractant Protein. The role ofthe inflammatory cytokines is to recruit the immune cells during theinfection [27].
Tobacco smoke contains nicotine which is a cytotoxic substance thatinduces immune-suppression, defective neutrophil functions andimpaired gingival health [28]. It was reported that cigarette smokesuppresses the activation of epithelial-mediated innate immuneresponses to bacterial infection.
The epithelium is a non-vascular tissue that depends on the adjacentconnective tissue. Inflammation modifies the blood availability andimpedes the elimination of metabolites from the epithelium [29]. Inthe current study, numerous capillaries and congestion of blood vesselswere noticed in group III. This was in accordance with some authors[13,26]. On the contrary, it was reported that moderate negativecorrelation was found between pack years and the number of bloodvessels [30]. It was also reported that decrease in capillary diameterand density of blood vessels were noticed in gingival tissues ofsmokers. This is sometimes confused with gingival health status asnoticed by reduction of tissue redness and bleeding upon probing [13].
In the current study, Mallory`s triple stain revealed disorganizationof collagen fibers and significant decrease in mean area percentage ofcollagen fibers in group III compared to control group. This wasexplained by the fact that cigarette smoke strongly increases cytokineswhich induce production of protease enzymes, including matrixmetalloproteinases (MMPs), elastase, and gelatinase, which destroy theextracellular matrix proteins (e.g. collagen, elastin, and gelatin) andcell-cell interaction proteins (e.g. E-cadherin) during the infection ortissue repair. It was also reported that inflammation causes connectivetissue disorganization [29].
Human gingival fibroblasts rapidly uptake and accumulate highlevels of nicotine in vitro, most of which remains inside the fibroblastswhere it can affect cell metabolism. In addition, nicotine inhibitsproliferation and migration of human periodontal ligament fibroblasts.Nicotine also acts on lysosomes by lowering the inner pH or byenlarging them [31]. Others reported that normal function of gingivalfibroblasts is essential for maintenance of the gingival extracellularmatrix, but under inflammation of the gingiva -which may occur withtobacco use-, they can also act in its destruction. Effect of tobacco ongingival fibroblast proliferation, production of fibronectin, productionand breakdown of type I collagen, was studied by some authors. Theyfound that nicotine at ≥ 0.025% significantly increased collagenaseactivity in vitro; inhibited growth of gingival fibroblasts and theirproduction of fibronectin and collagen and promoted collagenbreakdown. This suggested that nicotine itself might augment thedestruction of the gingival extracellular matrix [32].
On the contrary, greater collagenization was noticed in connectivetissue of smokers [33]. Clinically, it has been widely recognized that
Citation: Hamam GG, El-Waseef DAEA (2018) Effect of Cigarette Smoking on Human Gingival Mucosa-Histological and Morphometric Study. J CytolHistol 9: 517. doi:10.4172/2157-7099.1000517
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J Cytol Histol, an open access journalISSN: 2157-7099
Volume 9 • Issue 4 • 1000517
fibrosis is frequently observed in the smoker’s gingiva. Nicotineincreased the production of connective tissue growth factor protein ingingival fibroblasts and promoted periodontal fibrosis [29]. Thisdiscrepancy might be attributed to the fact that the effect of nicotineon fibroblast viability and growth, depends on the concentration aswell as duration of cell exposure [29].
In the current study, group III showed significant increase in themean area parentage of caspase-3 positive reaction in epithelial cellsand a significant decrease in the mean area percentage of collagenfibers in the lamina propria, compared to group II. It was reported thatthe amount and the duration of exposure to a carcinogen are oftencombined to give a single measure of lifetime cumulative dose [34].
There are controversial observations on the effect of smoking ongingival tissue. Some reports indicated that smokers had more gingivalinflammation; others observed less gingival inflammation in smokersthan non-smokers. In this study inflammation was more often seen insmokers. When collagen fibers were evaluated, we have also found thatthey decreased in lamina propria of smoker's gingiva. Others reportedincrease collagen fibers. This discrepancy could be attributed to smallernumber of samples in this study, as well as the fact that our sampleswere obtained from the gingiva of the lower jaw and it was reportedthat the impact of cigarette smoke is greater on the upper jawcompared to the lower as they were more exposed to tobacco smoke[33].
ConclusionThe present study indicated that despite the normal appearance of
gingiva in smokers, cigarette smoke could change the structure ofhuman gingival mucosa in a dose dependent manner.
RecommendationsA gingival biopsy every 6 month is recommended for Tobacco
smokers as a follow up, not only for early detection of any oralepithelial neoplasms, but also as a detector for the respiratory epithelialneoplasms which are more dangerous and more difficult to bebiopsied.
Tobacco smoking is a Public Health problem with serious sideeffects and it needs intensive and comprehensive tobacco controlefforts.
AcknowledgementsWe would like to thank Professor Dr. Hussein M. Taher and his
team for their kind helps in history and consent taking, in examinationand obtaining the specimens.
Ethical ApprovalAll procedures performed in studies involving human participants
were in accordance with the ethical standards of the institutional andnational research committee.
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Citation:Hamam GG, El-Waseef DAEA (2018) Effect of Cigarette Smoking on Human Gingival Mucosa-Histological and Morphometric Study. J CytolHistol 9: 517. doi:10.4172/2157-7099.1000517
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